Abstract Protein glycosylation is ubiquitously involved in all domains of life and holds the key to elucidating molecular mechanisms underpinning disease. Despite the biomedical and clinical significance of glycans, progress in glycomics has considerably lagged behind genomics and proteomics, owing to enormous structural diversity and dynamic alternation of glycans arising from complex non-template driven biosynthesis. Thus it is imperative to develop innovative technologies and instrumentation that can catalyze key breakthroughs in glycomic analysis. This proposal intends to address the urgent need of the next-generation glycomics technologies by leveraging on new microfluidic platforms stemming from research in the PI's laboratory. Specifically, the PI proposes to develop a novel microfluidic actuator-based single-molecule analysis method that can substantially improve the analytical performance of lectin microarray. Moreover, the PI seeks to integrate sample processing functionalities with the digital lectin-antibody barcode array system to construct a ?sample-in-answer-out? digital microfluidic lectin array platform for sensitive and quantitative targeted glycomic profiling in a high-throughput manner. The project consists of three aims: 1) develop a digital microfluidic approach for single-molecule quantification of low-abundance glycoproteins; 2) develop a fully integrated microsystem for multiplexed, quantitative targeted glycomic profiling; and 3) demonstrate quantitative comparative glycomic profiling of plasma from ovarian cancer patients. This research, if successful, will yield a key transformative tool for systems glycomics and allow the PI to pursue the long-term interest in biomedical investigation of pathological roles of protein glycosylation through obtaining dynamic overview of human plasma glycome.